This invention relates to chimeric proteins which include a cytokine and an enzymatically inactive polypeptide, and therapeutic uses thereof.
Cytokines have a wide range of effects on cell growth and differentiation. The value of certain cytokines has been recognized, including, for example, IL-2 for promoting the growth of activated T cells, B cells, LAK cells, and NK cells; IL-3 for promoting the growth of pluripotent hematopoietic progenitor cells; granulocyte macrophage-colony stimulating factor (GM-CSF) for promoting the growth and differentiation of neutrophils and macrophages, and for activating macrophages; kit ligand for promoting basophil and mast cell differentiation; IL-4 for promoting B cell proliferation, enhancing class II gene expression, enhancing IgG1 and IgE production, and promoting activated T cell proliferation and effector cell function; IL-5 for enhancing IgA production and stimulating eosinophil growth; IL-6 for transiently blocking myeloma growth, inducing immunoglobulin production, and inducing plasma cell and hepatocyte growth; IL-7 for inducing immature and mature B and T cell growth; and interferon-xcex1 and -xcex2 for their antiviral activity against papilloma viruses, hepatitis viruses, and herpes virus, and for treating hairy cell leukemia, myeloma, and other hematopoietic malignancies. Some additional functions of cytokines are summarized below.
Reported IL-1 activities include activation of T cells; induction of IL-2 receptor expression and cytokine gene expression; enhancement of collagenase, stromelysin, prostaglandin, and PDGF-AA synthesis by fibroblasts; co-stimulation of thymocyte proliferation; stimulation of pre-B cell differentiation; co-stimulation of B cell proliferation and Ig secretion; augmentation of IL-2 and IFN-induced activation of NK-mediated cytotoxicity; induction of adhesion molecule expression by endothelial cells; osteoblast and endothelial cell activation; enhancement of collagen production by epidermal cells; modulation of reparative functions following tissue injury; induction of insulin secretion; and xcex2-islet cell cytotoxicity.
IL-1 has also been shown to stimulate the release of factors associated with the growth and differentiation of cells from myeloid and lymphoid lineages in vitro. IL-1 is thought to induce the production of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) by human marrow stromal cells; induce the production of GM-CSF and G-CSF by human dermal fibroblasts; and induce the production of GM-CSF by human peripheral blood lymphocytes. IL-1 also stimulates hematopoiesis by up-regulating receptors for colony stimulating factors and inducing the proliferation of pluripotent progenitors in the bone marrow. IL-1 has been shown to protect mice from otherwise lethal doses of radiation which indicates that this protein is useful in cancer therapy. Also, IL-1 has been shown to accelerate wound healing, presumably due to its ability to induce angiogenesis and fibroblast activation.
IL-2 has been reported to participate in the activation, tumoricidal activity, and growth of T cells, NK cells, and LAK cells; augment B cell growth and immunoglobulin production; augment IFN-xcex3 production; induce IL-6 production by human monocytes; modulate histamine release by stimulated basophils; and modulate expression of the IL-2 receptor. Applications of IL-2 include anti-tumor therapy employing IL-2-activated LAK and TIL cell infusions; augmentation of IL-2 levels in treating immunodeficiency disorders, and increasing of NK cell activity following bone marrow transplant.
Reported functions of IL-3 include stimulating the proliferation of mast cell lines; stimulating the formation of neutrophils, macrophages, megakaryocytes, basophils, eosinophils, and mast cells from isolated hematopoietic progenitors; enhancing growth of certain human T lymphocytes; and potentiating the activity of eosinophils, basophils, and monocytes. It has been shown that IL-3 exerts its ability to support multi-lineage colony formation early in the development of multipotent progenitors. IL-3 exhibits synergy with Stem Cell Factor (kit ligand) in inducing human CD34+ cells to form basophils and mast cells. IL-3 has been used successfully in combination with factors such as GM-CSF to stimulate hematopoiesis in primates. In addition, sequential administration of IL-3 and IL-6 in primates stimulates thrombopoiesis. In vitro studies suggest that IL-3 can be used to reverse the hematopoietic toxicity associated with AZT treatment. Recombinant IL-3 has also been used in clinical trials in combination with other colony stimulating factors as a treatment for aplastic anemia.
IL-4 has been reported to be useful for up-regulating MHC Class II expression in resting B cells; enhancing IgG1, IgE, and sIgM production by B cells; up-regulating Fc receptor expression for IgE on B cells and monocytes; increasing viability and growth of normal resting cells and certain T cell lines; co-stimulating growth in certain mast cell lines; maintaining Lyt-2xe2x88x92/L3T431  thymic stem cells; promoting thymocyte-maturation; enhancing the proliferation of granulocyte-macrophage progenitors, erythrocyte progenitors, and meqakaryocytes in response to G-CSF, EPO, and IL-1, respectively; inhibiting human breast carcinoma cell growth in culture; inducing progression in B cells; inducing tumoricidal activity in cultured macrophages; and regulating adhesion molecule expression on endothelial cells.
Additionally, IL-4 is thought to act in combination with IL-1 as an autocrine growth factor for antigen-specific T cells to enhance antigen presentation and phagocytosis in macrophages. IL-4 not only enhances the development of cytotoxic T lymphocytes (CTL) from resting murine cells, but it also induces LAK activity. As a multifunctional cytokine that is reported to augment certain T and B cell responses, the therapeutic functions of IL-4 include reconstitution of cellular and humoral immune function following bone marrow transplantation; induction of terminal differentiation of acute lymphoblastoid leukemias; amelioration of immunodeficiency associated with hyper IgM; inhibition of the growth of solid tumors and B cell lymphomas; and reduction of inflammatory processes through down-regulation of production of IL-1, TNF, and IL-6. IL-4 has also been used in preclinical models to treat T cell-dependent autoimmune diseases, e.g., autoimmune diabetes mellitus and experimental and T cell-dependent allergic encephalomyelitis (Rapoport et al., 1993, J. Exp. Med. 178:87-99 and Racke et al., 1994, J. Exp. Med. 180:1961). Hence, IL-4 may be used in treating a variety of pathologies involving T cell-dependent immune activities.
IL-5 has been shown to induce eosinophil colonies in human liquid bone marrow cultures to induce antibody-mediated killing of tumor cells by peripheral blood eosinophils. IL-5 also has been shown to stimulate murine B cells to differentiate and proliferate and to simulate IgA and IgM secretion in B cells. IL-5 is useful for treating pathologies related to alterations in eosinophil activity. For example, suggested uses of IL-5 include treatment of schistosomiasis (see, e.g., Sanderson, April, 1989, xe2x80x9cInternational Conference on the Clinical Impact of Interleukinsxe2x80x9d at the Royal College of Physicians in London). Other reports suggest the use of IL-5 in treating patients having certain tumors (see, e.g., Kolb et al., 1979, Br. J. Cancer 40:410; Pretlow et al., 1983, Cancer Res. 43:2997; and Iwasaki et al., 1986, Cancer 58:1321).
IL-6 is reported to exhibit multiple functions, including induction of proliferation in a number of cells, including EBV-transformed B cells, T cells, mesangial cells, and keratinocytes; enhancement of the IL-3-dependent proliferation of multipotential hematopoietic progenitors; promotion of megakaryocyte maturation; triggering of neuronal differentiation; growth inhibition of certain melanoma cell lines, myeloid leukemic cell lines, and breast carcinoma cell lines; induction of B cell differentiation; stimulation of IgG secretion; and induction of cytotoxic T cell differentiation.
Additionally, IL-6 acts on murine thymocytes to induce the differentiation of Lyt-2+ CTL in the presence of IL-2, and IL-6 supports the proliferation of Con-A or T cell receptor antibody-stimulated T cells in vitro. IL-6 has also been reported to co-stimulate thymocyte proliferation and induce the release of acute phase reactants from hepatocytes. IL-6 is also thought to be an autocrine growth factor for tumor cells from patients with multiple myeloma.
IL-7 has been reported to have T cell growth factor activity. Stem cell factor synergizes with IL-7 to stimulate proliferation of early T cell progenitors. Also, IL-7 acts as a co-stimulus with Con A to induce the proliferation of purified murine T cells. IL-7 has also been reported to induce proliferation of human peripheral blood T lymphocytes in the presence of sub-mitogenic doses of Con A and PHA. Certain studies suggest that IL-7 acts directly on human CD8+ T cells to augment cytotoxicity and that IL-7 is a potent differentiation factor for the development of CTL.
In mice, IL-7 has been shown to act on CD8+ T cells to induce CTL in an IL-2- and IL-6-dependent manner. IL-7 is required for the IL-1-induced proliferation of murine thymocytes. IL-7 has further been shown to induce LAK activity from CD8+ cells prepared from murine peripheral lymphoid tissues. IL-7 has been shown to increase surface expression of the ICAM-1 molecule on melanocytes and melanoma cells. Injection of IL-7 into mice leads to a 3 to 5-fold increase in circulating immature B cells with a concurrent 90% reduction in myeloid progenitors in the bone marrow and a 15-fold increase in myeloid progenitors in the spleen. Hence, IL-7 is thought to have a similar spectrum of therapeutic activities in vivo as those reported for IL-2.
IL-9 stimulates the proliferation of mouse erythroid progenitors and promotes erythroid differentiation of cells in the presence of erythropoietin and IL-3 (Bourett et al., 1992, Exp. Hematol. 20:868). IL-9 has also been shown to enhance the survival of T cell lines in vitro (Renaud et al., 1990, Cytokine 2:9). IL-9 also potentiates IL-4-dependent Ig production by human B lymphocytes, and it promotes IL-6 production by murine mast cells lines derived from bone marrow. In addition, IL-9 is involved in the differentiation of hippocampal progenitors (Uyttenhove et al., 1991, J. Exp. Med. 173:519).
IL-10 is a cytokine produced by activated Th2 cells, B cells, keratinocytes, monocytes, and macrophages (Moore et al., 1993, Annu. Rev. Immunol. 11:165). IL-10 can be used to stimulate growth and differentiation of activated human B cells. In vitro, murine and human IL-10 inhibit cytokine synthesis (e.g., IFN-xcex3, TNF-xcex2, and IL-2) by Th1 cells, NK cells, monocytes, and macrophages (Fiorentino et al., 1989, J. Exp. Med., 170:2081-2095; Fiorentino et al., 1991, J. Immunol. 146:3444; Hsu et al., 1992, Int. Immunol. 4:563; Hsu et al., 1992, Int. Immunol. 4:563; D""Andrea et al., 1993, J. Exp. Med. 178:1041; and de Waal Malefyt et al., 1991, J. Exp. Med. 174:915; Fiorentino et al., 1991, J. Immunol. 147:3815). Thus IL-10 is useful for inhibiting Th1 responses to prevent transplant rejection and T cell-mediated autoimmune diseases, such as type I diabetes and multiple sclerosis. The ability of IL-10 to inhibit secretion of the pro-inflammatory cytokines (e.g., IL-1, IL-6, IL-8, and TNF-xcex1) suggests that IL-10 is a useful anti-inflammatory agent in the treatment of rheumatoid arthritis and psoriasis.
IL-10 has been recognized for its value in treating septicemia. Gram-negative septicemia in hospitalized patients is invariably associated with high morbidity and mortality (Bone, 1991, Ann. Intern. Med. 115:457). Case fatality rates of 20-60% reflect the frequent development of acute lung injury (Byrne et al., 1987, Acute Care 13:206) and multiple organ failure (Abrams et al., 1989, Surg. Rounds 12:44), as well as the lack of effective therapies. Endotoxin (LPS)., a product of gram-negative bacteria, is a major causative agent in the pathogenesis of septic shock (Glausner et al., 1991, Lancet 338:732). A septic shock-like syndrome can be induced experimentally by a single injection of LPS into animals. Injection of IL-10 into mice inhibits secretion of tumor necrosis factor in vivo and protects against the lethal effects of endotoxin (Gerard et al., 1993, J. Exp. Med. 177(2):547; de Waal Malefyt et al., 1991, J. Exp. Med. 174:915; Fiorentino et al., 1991, J. Immunol. 147:3815; and Moore et al., 1990, Science 248:1230).
Upon infection with Schistosoma, a genus of flatworms, the organism deposits its eggs into the liver, causing granuloma formation and fibrosis of liver tissue. Liver damage caused by Schistosome infection can lead to cirrhosis of the liver. Schistosomiasis is often chronic and debilitating.
Naturally-occurring cytokines have short circulating half-lives; for example, naturally-occurring IL-10 is therapeutically effective for approximately 30 minutes following administration (Gerard et al., 1993, J. Exp. Med. 177(2):547).
I have discovered that the in vivo half-life of a cytokine can be increased by bonding the cytokine to a polypeptide which increases the longevity of the cytokine while being enzymatically inactive in humans, and I have discovered that certain of the chimeric cytokines (i.e., chimeric proteins or chimeras) are useful for treating or inhibiting the onset of conditions such as septic shock, granulomatous disorders (e.g., schistosomiasis), Type I diabetes, certain cancers (e.g., multiple myeloma), and chronic infections.
Accordingly, in one aspect, the invention features a chimeric protein having a cytokine bonded to a polypeptide which is enzymatically inactive in humans and which increases the circulating half-life of the cytokine in vivo by a factor of at least 2, and preferably by a factor of at least 10.
Useful enzymatically inactive polypeptides include not only proteins that are not enzymes, such as albumin, but also enzymes that have enzymatic activity in an organism other than humans but which are inactive in humans. For example, useful polypeptides include plant enzymes, porcine or rodent glycosyltransferases, and xcex1-1,3-galactosyltransferases (see, e.g., Sandrin et al., 1993, PNAS 90:11391).
The enzymatically inactive polypeptide can include an IgG hinge region and a half-life increasing polypeptide. In this embodiment, the IgG hinge region is bonded to the cytokine and serves as a flexible polypeptide spacer between the cytokine and the half-life-increasing polypeptide (e.g., IgG Fc or albumin).
When the enzymatically inactive polypeptide includes an IgG hinge region and the Fc region of an IgG molecule, it lacks an IgG variable region of a heavy chain so that the binding specificity conferred by the variable region is lacking in the chimera. The Fc region can include a mutation which inhibits complement fixation and prevents Fc from binding the Fc receptor with high affinity, thus preventing the chimera from being lytic. Alternatively, the Fc region can be lytic, i.e., be able to bind complement and bring about lysis of the cell to which the chimera binds.
The cytokine portion of the chimeric protein can be an interleukin, such as IL-10, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-12, or IL-15. Other useful cytokines include GM-CSF, G-CSF, interferons (e.g., IFN-xcex1, IFN-xcex2, and IFN-xcex3), and tumor necrosis factors (e.g., TNF-xcex1 and TNF-xcex2).
A chimeric cytokine of the invention can be used in a therapeutic composition formed by admixture of the chimeric protein with a pharmaceutically acceptable carrier. In various embodiments, the invention provides methods for treating or inhibiting the development of a variety of conditions. For example, the IL-10 chimeras and IL-4 chimeras can each be used to treat or inhibit granuloma formation (e.g., schistosomiasis). IL-10 chimeras and IL-4 chimeras are also useful for inhibiting the development of Type I diabetes, for treating or inhibiting the development of Crohn""s disease, ulcerative colitis, or Boeck""s disease. The IL-10 chimera is useful for treating or inhibiting septic shock. IL-6 chimeras are useful for treating or inhibiting the development of multiple myeloma in a patient. TNF-xcex1 chimeras and TNF-xcex2 chimeras each are useful for combatting cervical cancer caused by papilloma viruses, liver cancer caused by hepatitis viruses, and skin eruptions caused by herpes viruses.
By xe2x80x9ccytokinexe2x80x9d is meant any of the non-antibody proteins released by one cell population (e.g., primed T-lymphocytes) on contact with specific antigen, which act as intercellular mediators, as in the generation of an immune response. One important class of cytokines are those which induce proliferation of lymphocytes, e.g., T cells.
By IgG xe2x80x9cFcxe2x80x9d region is meant a naturally-occurring or synthetic polypeptide homologous to the IgG C-terminal domain that is produced upon papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kD. In the molecules of the invention, the entire Fc region can be used, or only a half-life enhancing portion. In addition, many modifications in amino acid sequence are acceptable, as native activity is not in all cases necessary or desired.
By xe2x80x9cnon-lyticxe2x80x9d IgG Fc is meant an IgG Fc region which lacks a high affinity Fc receptor binding site and which lacks a Cxe2x80x21q binding site. The high affinity Fc receptor binding site includes the Leu residue at-position 235 of IgG Fc; the Fc receptor binding site can be functionally destroyed by mutating or deleting Leu 235. For example, substitution of Glu for Leu 235 inhibits the ability of the Fc region to bind the high affinity Fc receptor. The Cxe2x80x21q binding site can be functionally destroyed by mutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG1. For example, substitution of Ala residues for Glu 318, Lys 320, and Lys 322 renders IgG1 Fc unable to direct ADCC.
By xe2x80x9clyticxe2x80x9d IgG Fc is meant an IgG Fc region which has a high affinity Fc receptor binding site and a Cxe2x80x21q binding site. The high affinity Fc receptor binding site includes the Leu residue at position 235 of the IgG Fc. The Cxe2x80x21q binding site includes the Glu 318, Lys 320, and Lys 322 residues of IgG1. Lytic IgG Fc has wild-type residues or conservative amino acid substitutions at these binding sites. Lytic IgG Fc can target cells for antibody dependent cellular cytotoxicity (ADCC) or complement directed cytolysis (CDC).
By IgG xe2x80x9chingexe2x80x9d region is meant a polypeptide homologous to the portion of a naturally-occurring IgG which includes the cysteine residues at which the disulfide bonds linking the two heavy chains of the immunoglobulin form. For IgG1, the hinge region also includes the cysteine residues at which the disulfide bonds linking the xcex31 and light chains form. The hinge region is approximately 13-18 amino acids in length in IgG1, IgG2, and IGg4; in IgG3, the hinge region is approximately 65 amino acids in length.
By polypeptide xe2x80x9cspacerxe2x80x9d is meant a polypeptide which, when placed between the half-life-increasing polypeptide and a cytokine, possesses an amino acid residue with a normalized B value (Bnorm; a measure of flexibility) of 1.000 or greater, preferably of 1.125 or greater, and, most preferably of 1.135 or greater (see, e.g., Karplus et al., 1985, Naturwissenschaften 72:212). Amino acids which are commonly known to be flexible include glutamic acid, glutamine, threonine, lysine, serine, glycine, proline, aspartic acid, asparagine, and arginine.
The invention offers several features and advantages: (1) the chimeric proteins of the invention have an extended circulating half-life and provide long term protection; (2) because many of the cytokines and longevity-increasing polypeptides useful in the invention have already been purified, the chimeric proteins can easily be purified by employing methods that have been described for purifying the cytokine or longevity-increasing polypeptide; (3) some of the chimeric proteins are mutated such that they are defective for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement directed cytolysis (CDC), thus making them useful for treating or inhibiting the onset of septic shock, type I diabetes, or multiple myeloma without destroying the target cells.
An additional advantage of chimeric proteins that include an Fc polypeptide is that they cannot cross the blood/brain barrier and enter the brain where polypeptides such as IL-6, tumor necrosis factor, IL-1xcex1, and IL-1xcex2 could cause side effects by reacting with-regulatory centers in the brain. Among the side effects caused by these cytokines in the absence of an Fc polypeptide are somnolence, fever, and hypotension.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.